Journal of Ornithology - University of South Carolina
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Journal of Ornithology - University of South Carolina
Journal of Ornithology Ecological differences in response of bird species to radioactivity from Chernobyl and Fukushima --Manuscript Draft-Manuscript Number: JORN-D-14-00257R2 Full Title: Ecological differences in response of bird species to radioactivity from Chernobyl and Fukushima Article Type: IOC2010-Proceedings Keywords: Birds Chernobyl Coloration Fukushima Pigments Radiation resistance Corresponding Author: Anders Pape Moller FRANCE Corresponding Author Secondary Information: Corresponding Author's Institution: Corresponding Author's Secondary Institution: First Author: Anders Pape Moller First Author Secondary Information: Order of Authors: Anders Pape Moller Timothy A. Mousseau Isao Nishiumi Keisuke Ueda Order of Authors Secondary Information: Abstract: Organisms differ in their susceptibility to ionizing radiation although the ecological basis for such differences remain poorly understood. We hypothesized that ecological characteristics such as body size, diet, migration and pigments of plumage would predict the impact of radiation on abundance through effects on relative food consumption rates, free radicals and antioxidants. We made 2398 breeding bird censuses and quantified the impact of radiation on abundance at Chernobyl and Fukushima providing statistical replication, but also analyses of interaction effects. The impact of radiation on abundance of birds was stronger in Fukushima than in Chernobyl. Species with small body size and hence relatively high food consumption rates were more negatively impacted. Secondary consumers showed stronger negative effects of radiation on abundance than herbivores, especially in Fukushima. There was no main effect of migration, but migrants were more negatively impacted in Chernobyl, while residents were more negatively impacted in Fukushima. Carotenoid and pheomelanin plumage pigments associated with antioxidant status showed stronger negative effects, especially in Chernobyl compared to Fukushima, while eumelanic coloration that is not related to antioxidant status did not show such an effect. These differences between Chernobyl and Fukuskima may reflect differences in duration of exposure, differences in radioactive isotopes and differences in accumulation of mutations. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation Manuscript Click here to download Manuscript: Radiation-Census-Review-NoTrackChanges.doc Click here to view linked References 1 1 Ecological differences in response of bird species to 2 radioactivity from Chernobyl and Fukushima 3 A. P. Møller1, T. A. Mousseau2, I. Nishiumi3, K. Ueda4 4 5 1 6 Laboratoire d'Ecologie, Systématique et Evolution, CNRS UMR 8079, Université Paris-Sud, Bâtiment 362, F-91405 Orsay Cedex, France; 7 2 8 Department of Biological Sciences and the Environment and 9 Sustainability Program, University of South Carolina, Columbia, SC 10 29208, USA; 3 11 4-1-1 Amakubo, Tsukuba, Ibaraki 305-0005, Japan; 12 13 14 Department of Zoology, National Museum of Nature and Science, 4 Department of Life Science, Rikkyo University, 3-34-1 Nishi-ikebukuro, Toshima-ku, Tokyo 171-8501, Japan 15 16 17 Word count: 6339 18 Electronic supplementary material: 1760 19 20 Correspondence to APM: 21 Tel: (+33) 1 69 15 56 88 22 Fax: (+33) 1 69 15 56 96 23 E-mail: [email protected], [email protected], 24 [email protected], [email protected] 25 26 2 27 Abstract Organisms differ in their susceptibility to ionizing radiation 28 although the ecological basis for such differences remain poorly 29 understood. We hypothesized that ecological characteristics such as body 30 size, diet, migration and pigments of plumage would predict the impact of 31 radiation on abundance through effects on relative food consumption rates, 32 free radicals and antioxidants. We made 2398 breeding bird censuses and 33 quantified the impact of radiation on abundance at Chernobyl and 34 Fukushima providing statistical replication, but also analyses of interaction 35 effects. The impact of radiation on abundance of birds was stronger in 36 Fukushima than in Chernobyl. Species with small body size and hence 37 relatively high food consumption rates were more negatively impacted. 38 Secondary consumers showed stronger negative effects of radiation on 39 abundance than herbivores, especially in Fukushima. There was no main 40 effect of migration, but migrants were more negatively impacted in 41 Chernobyl, while residents were more negatively impacted in Fukushima. 42 Carotenoid and pheomelanin plumage pigments associated with antioxidant 43 status showed stronger negative effects, especially in Chernobyl compared 44 to Fukushima, while eumelanic coloration that is not related to antioxidant 45 status did not show such an effect. These differences between Chernobyl 46 and Fukuskima may reflect differences in duration of exposure, differences 47 in radioactive isotopes and differences in accumulation of mutations. 48 49 Keywords Birds Chernobyl Coloration Fukushima Pigments 50 Radiation resistance 51 52 3 53 Introduction 54 There is considerable interspecific variation among organisms in 55 susceptibility to radiation effects (e. g. Asker et al. 2011; Romanovskaya et 56 al. 2002; Dighton et al. 2008; Møller and Mousseau 2013). Why that is the 57 case remains an open question. This may arise from differences in exposure 58 and hence differences in the intensity of natural selection that have resulted 59 in the evolution of mechanisms that sustain or even nullify such effects of 60 ionizing radiation. This interesting scientific question has received little 61 attention despite the fact that many species are tolerant to extreme levels of 62 radiation (review in Møller and Mousseau 2013). A few studies that have 63 attempted to assess interspecific differences in susceptibility to radiation 64 and interspecific differences in their ecological correlates (Møller and 65 Mousseau 2007b, 2011b; Galván et al. 2011, 2014). Ionizing radiation 66 produces free radicals and thus promotes the generation of reactive 67 oxidative species that deplete antioxidant levels in animals (Riley 1994; 68 Ivaniota et al. 1998; Neyfakh et al. 1998). Not surprisingly, interspecific 69 variation in susceptibility to radiation can be linked directly to ecological 70 traits that are associated with high rates of use of antioxidants such as long 71 distance migration, dispersal, allocation of antioxidants to coloration and 72 allocation of antioxidants by mothers to their offspring (Møller and 73 Mousseau 2007b). Field studies of individual birds in the vicinity of 74 Chernobyl have shown increased rates of DNA damage at higher levels of 75 background radiation (Bonisoli-Alquati et al. 2010b), and this effect can be 76 linked directly to an increased level of oxidative stress caused by 77 background radiation (Bonisoli-Alquati et al. 2010a). 78 Among the pigments responsible for integument color, melanins 79 and carotenoids are some of the most prominent and widespread. 80 Phaeomelanin- and carotenoid-based colors share a strong dependence on 81 antioxidant status. Vertebrates synthesize two main forms of melanin: 4 82 eumelanin and pheomelanin, with the latter producing lighter colors than 83 the former. Glutathione (GSH) levels determine the melanin form that is 84 synthesized, as melanogenesis can either lead to the production of 85 eumelanin when the activity of tyrosinase is high and the ratio of 86 cysteine:dopaquinone is low, or to the production of pheomelanin or even 87 an absence of melanin synthesis under opposite conditions (Ozeki et al. 88 1997; Galván and Alonso-Alvarez 2009). Therefore, eumelanogenesis 89 takes place when the levels of GSH are low and pheomelanogenesis when 90 the levels of GSH are high, implying that pheomelanogenesis proceeds 91 with higher levels of endogenous oxidative stress as compared to 92 eumelanogenesis (Galván and Solano 2009). The evolutionary implication 93 of this mechanism is that species in which natural selection has favored the 94 development of pheomelanic traits may have a decreased capacity to 95 combat oxidative stress as compared to species with eumelanic traits, as the 96 maintenance of high GSH levels as required by pheomelanogenesis might 97 be metabolically costly under adverse environmental conditions that 98 generate oxidative stress and thus consume GSH resources (Galván and 99 Alonso-Alvarez 2009; Galván and Solano 2009). 100 GSH is one of the antioxidants most susceptible to radiation (Bump 101 and Brown 1990; Navarro et al. 1997; Vartanian et al. 2004). Therefore, 102 species that have the molecular basis to produce large amounts of 103 pheomelanin may be more limited in their use of GSH to combat oxidative 104 stress, and may thus be more susceptible to ionizing radiation than species 105 in which melanogenesis has been selected to be directed toward the 106 production of eumelanin (Galván et al. 2011). Adaptation to radiation in 107 radioactively contaminated areas and naturally contaminated areas alike 108 may result in increased glutathione levels and body condition, while 109 oxidative stress and DNA damage has decreased with increasing 110 background radiation (Galván et al. 2014). This apparent adaptation to 5 111 radiation appears to be linked to larger mounts of pheomelanin and smaller 112 amounts of eumelanin being costly as shown from decreased glutathione 113 levels, increased oxidative stress and DNA damage, and reduced body 114 condition (Galván et al. 2014). Finally pheomelanin has been shown to 115 induce a change towards the production of less pro-oxidant forms of 116 pheomelanin that may help acclimatize birds to radiation exposure (Galván 117 et al. 2014). Indeed levels of gluthathione and pheomelanin are 118 significantly correlated (Galván et al. 2014). 119 Here we report the results of analyses of unique data on the 120 abundance of breeding birds in Fukushima, Japan and Chernobyl, Ukraine 121 in relation to background levels of radiation. There has been accumulation 122 of mutations in Chernobyl since 1986 for more than 20 generations for 123 many small birds that typically have very short lifespan (Møller and 124 Mousseau 2011b), while only a couple of generations have passed since the 125 accident in 2011 in Fukushima. Thus, we predicted significant differences 126 in the association between abundance and radiation between Chernobyl and 127 Fukushima for different species of birds implying a significant area by 128 ecological variable interaction. 129 The objectives of this study were to test (1) whether the effect of 130 radiation reduced the abundance of birds; (2) whether this effect differed 131 among species; (3) whether body mass, diet, migration and pigment-based 132 coloration affected the abundance of birds; and (4) whether the effects of 133 these predictors differed between Fukushima and Chernobyl, as predicted 134 from their different history of exposure to radioactive contaminants. Small- 135 sized species have relatively higher metabolism, higher intake rates and 136 hence higher ingestion of radionuclides. Similarly, species at higher trophic 137 levels should suffer more from radioactive contamination as a result of 138 bioaccumulation. Migratory birds through their high metabolic rate and use 139 of antioxidants should differ in impact of radiation compared to residents. 6 140 Indeed, negative impacts of radiation on abundance are stronger on 141 abundance of migrants than resident species (Møller and Mousseau 2007b). 142 This is contrary to expectation because migrants spend less time in the 143 contaminated breeding areas than residents. However, migrants deplete 144 their stores of antioxidants during migration resulting in very low levels 145 upon arrival (Ninni et al. 2004). This effect is detrimental because breeding 146 birds start establishing territories, nest building and egg formation upon 147 arrival causing antioxidant levels to be severely depleted (Møller et al. 148 2005). Finally, there is a trade-off between allocation of antioxidants to 149 production of feather color and self-maintenance in birds with carotenoid- 150 and pheomelanin-based plumage. Therefore, any individual that allocated a 151 large fraction of antioxidant resources to plumage coloration without 152 consideration of future uses for protection against oxidative stress caused 153 by radiation would be at a selective disadvantage. Likewise, such 154 individuals would also risk increased rates of mutations due to the lack of 155 protection against oxidative stress thus reducing survival prospects. 156 157 Methods 158 Study sites 159 The breeding bird census points were located at ca. 100 m intervals in 160 forested areas west of the exclusion zone around the Fukushima Daiichi 161 power plants in 2011-2014 (Fig. 1A) or in forested areas within the 162 Chernobyl Exclusion Zone or adjacent areas, or in areas in southern 163 Belarus around Gomel during the breeding seasons 2006-2009 (Fig. 1B). 164 At least one local ornithologist participated in the censuses in Japan to 165 confirm the identity of some difficult bird species. 166 167 Census methods 7 168 We adopted the point count census method, which provides reliable 169 information on relative abundance of birds (Blondel et al. 1970; Møller 170 1983; Bibby et al. 2005; Voříšek et al. 2010). The method is based on an 171 observer recording for a period of five minutes all birds seen and heard. 172 Extensive national monitoring programs for breeding and wintering birds 173 based on point counts occur in many different countries, and this effort is 174 part of environmental monitoring by the European Union (Voříšek et al. 175 2010). This method has provided highly repeatable results for birds and 176 other animals at Chernobyl (Møller and Mousseau 2011a). It consists of 177 counts lasting 5 minutes during which the number of birds seen or heard is 178 recorded. APM conducted these standard point counts during 29 May – 9 179 June 2006, 1 – 11 June 2007, 29 May – 5 June 2008, 1 – 6 June 2009 in the 180 surroundings of Chernobyl (898 census points) and during 11 – 15 July 181 2011, 14-19 2012, 14-19 2013 and 11-16 2014 in Fukushima (1500 census 182 points). Thus one single five minutes count was recorded for each point in 183 each of the study years. The fact that one person made all counts eliminates 184 any variance in results due to inter-observer variability. There are no bird 185 census data from Chernobyl or Fukushima before the accidents, nor have 186 other scientists to the best of our knowledge conducted bird censuses 187 comparable to ours in the years following the accidents. 188 We directly tested the reliability of our counts by letting two persons 189 independently perform counts, and the degree of consistency was high for 190 both species richness, total abundance and abundance of individual species 191 (details reported by Møller and Mousseau 2007a for Chernobyl; similar 192 results exist for Fukushima (A. P. Møller, I. Nishiumi and T. A. Mousseau 193 unpublished data)). 194 Abundance estimates can be affected by numerous confounding 195 variables (Voříšek et al. 2010), and, therefore, it is important to control 196 such variables statistically to assess the underlying relationship between 8 197 radiation and species richness and abundance. We classified habitats 198 (agricultural habitats with grassland or shrub, deciduous forest, or 199 coniferous forest) and estimated to the nearest 10% ground cover by herbs, 200 shrub, trees, agricultural habitat, deciduous forest and coniferous forest 201 within a distance of 50 m from the census points. We recorded altitude to 202 the nearest foot, using GPS. Weather conditions can affect animal activity 203 and hence census results (Voříšek et al. 2010), and we recorded cloud 204 cover at the start of each point count (to the nearest eighth), temperature 205 (degrees Celsius), and wind force (Beaufort). For each census point we 206 recorded time of day when the count was started (to the nearest minute). 207 Because activity may show a curvilinear relationship with time of day, with 208 high levels of bird activity in the morning and to a lesser extent in the 209 evening (Voříšek et al. 2010), we also included time squared as an 210 explanatory variable. 211 212 Background radiation 213 We measured radiation in the field and cross-validated these measurements 214 with those reported by the Ukrainian Ministry of Emergencies. Once 215 having finished the 5 minutes census we measured , , and radiation 216 levels at ground level directly in the field at each point where were 217 censused invertebrates using a hand-held dosimeter (Model: Inspector, SE 218 International, Inc., Summertown, TN, USA). We measured levels two-three 219 times at each site and averaged the results. We cross-validated our 220 measurements in Ukraine against data from the governmental 221 measurements published by Shestopalov (1996), estimated as the mid-point 222 of the ranges published. This analysis revealed a very strong positive 223 relationship (linear regression on log-log transformed data: F = 1546.49, df 224 = 1, 252, r2 = 0.86, P < 0.0001, slope (SE) = 1.28 (0.10)), suggesting that 9 225 our field estimates of radiation provided reliable measurements of levels of 226 radiation among sites. 227 In Fukushima we used the same dosimeters, and the measurements 228 were cross-validated with readings with a dosimeter that had been recently 229 calibrated and certified to be accurate by the factory (International 230 Medcom, Sebastopol, CA, USA). We cross-validated tests at Fukushima by 231 comparing our own measurements with measurements obtained at the same 232 locations with a TCS 171-ALOKA used by Japanese authorities. Again, 233 there was a very strong positive relationship (linear regression on log-log 234 transformed data: F = 2427.97, df = 1, 20, r2 = 0.99, P < 0.0001, slope (SE) 235 = 1.120 (0.023)). We have made extensive measurements of internal dose 236 in hundreds of birds in Chernobyl and found very strong positive 237 correlations between internal dose and background radiation level (A. P. 238 Møller and T. A. Mousseau unpublished manuscript). 239 240 Ecological variables 241 Body mass 242 We extracted mean body mass of males and females during the breeding 243 season from Cramp and Simmons (1977-1994), Lislevand et al. (2007) and 244 del Hoyo et al. (1995-2011). Body mass was estimated as the mean value 245 of the means for males and females. 246 247 Diet 248 We scored the diet of different species on a two level classification based 249 on information in Cramp and Simmons (1977-1994) and del Hoyo et al. 250 (1995-2011): (0) mainly plant material, especially outside the breeding 251 season; and (1) primary predators eating herbivores such as insects and 252 spiders, but also vertebrates. 253 10 254 Migration distance 255 We estimated migration distance as the difference in latitude between the 256 mean of the northernmost and the southernmost breeding distribution and 257 the mean of the northernmost and the southernmost winter distribution, 258 relying on information in Cramp and Perrins (1977-1994) and del Hoyo et 259 al. (1995-2011). A strict classification of species as either residents or 260 migrants resulted in similar conclusions (unpubl. data)). 261 262 Carotenoid-based coloration 263 We only considered pigment-based coloration in this study because we did 264 not have information on structural color for the species considered. The 265 extent of plumage patches colored by carotenoids was determined by 266 examining illustrations of adult breeding birds in Cramp and Simmons 267 (1977-1994) and Wild Bird Society of Japan (1982). We considered colors 268 that were yellow, orange and red to be caused by carotenoids (Tella et al. 269 2004; Olson and Owens 2005). We assigned scores that ranged from 0 270 (total lack of carotenoid-based color) to 5 (all carotenoid-based). 271 272 Melanin-based coloration 273 We used a method for assigning coloration to melanin developed by 274 (Galván et al. 2011). Information on melanin-based plumage coloration 275 was obtained by examining illustrations of bird species censused in 276 Chernobyl and Fukushima in Cramp and Simmons (1977-1994) and Wild 277 Bird Society of Japan (1982). Eumelanic and pheomelanic traits are 278 generally of distinctive colors, the former being responsible for black and 279 grey colors and the latter for yellowish, reddish, chestnut and brown colors 280 (Toral et al. 2008). Eumelanin and pheomelanin normally occur 281 simultaneously in tissues (Ozeki et al. 1997), but the darker colors 282 conferred by eumelanin (Toral et al. 2008) make evident the lower content 11 283 of this pigment in chestnut and brown as compared to black and grey colors 284 (Galván and Alonso-Alvarez 2009). Furthermore, many bird species have 285 feather melanin contents of high purity (> 90% of either eumelanin or 286 pheomelanin; McGraw and Wakamatsu 2004; J. J. Negro pers. com.). 287 Therefore, we considered that black and grey plumage colors were 288 predominantly generated by eumelanin, while chestnut and brown colors 289 were predominantly generated by pheomelanin. We did not consider 290 conspicuous yellow or red colorations assumed to be generated by other 291 pigments (i.e. carotenoids), unless chemically identified as melanin-based 292 by Toral et al. (2008). Although a rough approximation to the real 293 proportion of eumelanic and pheomelanic plumage, the assumption that 294 black-grey colors are eumelanic and brown-chestnut colors are 295 pheomelanic should be adequate for comparative purposes (Owens and 296 Hartley 1998). Thus, we quantified the proportion of melanic plumage 297 parts by examining illustrations in Cramp and Simmons (1977-1994) and 298 and Wild Bird Society of Japan (1982). Illustrations of both resting and 299 flying adult birds in breeding plumage were examined. The method used by 300 Beauchamp and Heeb (2001) and Galván (2008) was followed to obtain 301 estimates of the proportion of eu- and pheomelanic color present in the 302 plumage of each species, assigning scores that ranged from 0 (total lack of 303 melanic color) to 5 (all melanic). When a species was sexually dichromatic 304 regarding melanin-based coloration, eumelanic and pheomelanic scores 305 were the average obtained for males and females. When a species had 306 different subspecies or color morphs differing in extent or type of melanin- 307 based coloration, we used the nominate subspecies or the most common 308 morph, respectively. It must be noted that eu- and pheomelanic color 309 patches can coexist in the same feathers, and thus the sum of both color 310 scores, in a species that presents both color types, is not always necessarily 311 5, but higher values are also possible. 12 312 313 All interspecific data are reported in Appendix 1 in the electronic supplementary material. 314 315 Statistical analyses 316 Body mass, migration distance and radiation level were log10-transformed 317 and coverage with agricultural land, herbs, shrub and trees, deciduous 318 forest, coniferous forest and cloud cover were square-root arcsine- 319 transformed before analyses. 320 We quantified the relationship between abundance of different 321 species and level of radiation by estimating the Kendall rank-order 322 correlation for the relationship between abundance and log10-transformed 323 radiation while including all potentially confounding variables in the 324 statistical models (coverage by herbs, shrub, trees, agricultural habitat, 325 deciduous forest and coniferous forest, altitude, cloud cover, temperature, 326 wind force, time of day and time of day squared). We used non-parametric 327 rank order correlations because the assumptions of parametric analyses 328 were violated. We have previously shown that statistical control for the 329 confounding variables listed above did not affect the conclusions of 330 analyses (Møller and Mousseau 2011a). Therefore, we used the Kendall 331 rank-order correlations from analyses of abundance in relation to radiation 332 rather than partial rank-order correlations in the subsequent analysis. In this 333 model we included slope as the response variable and body mass, diet, 334 migration distance and area and the two-way interactions between body 335 mass, diet, migration distance and area as predictors. We did not include 336 other interactions because there were no a priori predictions. 337 It is a common underlying assumption of most statistical analyses 338 that each data point provides equally precise information about the 339 deterministic part of total process variation, i.e. the standard deviation of 340 the error term is constant over all values of the predictor variable(s) (Sokal 13 341 and Rohlf 1995). Because estimates of slopes depend on sample sizes, and 342 because sample sizes vary considerably among bird species, such variation 343 can have serious consequences for conclusions (Garamszegi and Møller 344 2010, 2011). The standard solution to this problem is to weight each 345 observation by sampling effort in order to use all data relative to their 346 importance, by giving each datum a weight that reflects its degree of 347 precision due to sampling effort (Draper and Smith 1981; Neter et al. 1996; 348 Garamszegi and Møller 2010). Therefore, we weighted the statistical model 349 by sample size. All analyses were made with JMP (SAS 2012). 350 351 Results 352 We investigated the impact of low-dose radiation on the abundance of 154 353 species of birds using 2398 standardized point counts, which resulted in 354 records of 15,811 individual birds. A total of 14 species are common to 355 Chernobyl and Fukushima allowing for comparison of the effect of 356 radiation on abundance of the same species in two different environments 357 (Møller et al. 2012a). The three species showing the strongest negative 358 correlated with level of background radiation level in Chernobyl were 359 yellowhammer Emberiza citrinella, chaffinch Fringilla coelebs and 360 blackcap Sylvia atricapilla. In Fukushima they were tree sparrow Passer 361 montanus, carrion crow Corvus corone and barn swallow Hirundo rustica. 362 The mean Kendall rank-order correlation between abundance and radiation 363 was -0.109 (SE = 0.012), N = 154 species, differing significantly from the 364 expected value zero in the absence of any effect of radiation (t = 9.18, df = 365 153, P < 0.0001). The mean correlation was -0.047 (0.008) in Chernobyl, 366 but almost three-fold stronger at -0.150 (SE = 0.022) in Fukushima, with 367 both the mean and the variance being significantly larger in Fukushima 368 than in Chernobyl (Welch ANOVA: F = 19.04, df = 1, 11735, P < 0.0001; 369 Levene’s test, F = 21.73, df = 1, 152, P < 0.0001). 14 370 The statistical model explaining variation in the correlations 371 between abundance and background radiation for different species 372 accounted for 64% of the variance (Table 1). Five out of seven main effects 373 were significant as were five out of six interaction effects between area and 374 ecological variables (Table 1). The correlations between abundance and 375 radiation level were more negative in species with smaller body mass, and 376 this effect differed significantly between Chernobyl and Fukushima as 377 shown by the significant interaction (Fig. 2; Table 1). Carnivorous species 378 showed more strongly negative correlations than herbivorous species and 379 this effect differed between areas with a larger difference in Fukushima 380 than in Chernobyl (Fig. 3; Table 1). Migration distance did not show a 381 significant main effect, but the migration effect differed between 382 Chernobyl and Fukushima with a stronger negative effect in migrants in 383 Chernobyl, and a stronger negative effect in residents in Fukushima (Fig. 4; 384 Table 1). 385 Plumage color pigments showed different relationships with impact 386 of radiation on abundance. Species with carotenoid-based coloration 387 showed a weaker negative effect than species without carotenoid-based 388 coloration (Table 1). The interaction between carotenoids and area was 389 significant with a stronger negative effect in species with than without 390 carotenoid-based plumage in Chernobyl, and a weaker negative effect in 391 species with than without such plumage in Fukushima (Fig. 5A). There was 392 only a weak and non-significant main effect of eumelanin and no 393 significant effect of the interaction (Fig. 5B, Table 1). There was no 394 significant main effect of pheomelanin (Table 1). A strong interaction 395 between pheomelanin and area implies that in Chernobyl species with 396 pheomelanin-based coloration experienced stronger negative effects of 397 radiation than species without such coloration, while in Fukushima species 15 398 with pheomelanin showed weaker negative effects than species without 399 pheomelanin in their plumage (Fig. 5C). 400 401 Discussion 402 The main findings of this extensive study of breeding birds at Chernobyl 403 and Fukushima were a negative impact of ionizing radiation on abundance 404 and a number of ecological characteristics explaining variation in 405 heterogeneity of this effect. We found significant main effects of body 406 mass, diet, migration and feather pigments on the impact of radiation on 407 abundance. Importantly, there were significant interactions between area 408 and ecological characteristics, implying that the effects of radiation were 409 not the same at Chernobyl and Fukushima. We will briefly discuss these 410 findings. 411 Body size is an important correlate of life history, physiology and 412 ecology (Bennett and Owens 2002). Small species eat disproportionately 413 more for their body size than large species due to their surface to volume 414 ratio. Therefore, we should expect small species to ingest 415 disproportionately more radionuclides than large species. Among bird 416 species in Fukushima there was a stronger negative relationship between 417 abundance and radiation in small-bodied species than among large species, 418 and a similar relationship was found in Chernobyl with a significant 419 interaction between radiation and area. Higher metabolism and higher rates 420 of ingestion by small species should also result in greater production of free 421 radicals and hence greater competition for anti-oxidants between use for 422 metabolism and use due to radiation exposure. 423 Diet and its interaction with study area was a significant predictor of 424 impact of radiation on abundance. We predicted and found an effect of 425 bioaccumulation of radionuclides in the food web (Voitovich and Afonin 426 2002; Yakushev et al. 1999). Animals at higher trophic levels generally 16 427 have higher levels of radionuclide concentrations than animals at lower 428 levels (e. g. Kryshev and Ryabov 1990; Kryshev et al. 1992; Smith et al. 429 2002). Here we found a stronger negative effect in secondary consumers 430 compared to herbivores, and the interaction between diet and area was 431 statistically significant, with a stronger effect in Fukushima compared to 432 Chernobyl. 433 Long-distance migration involves high metabolic effort and 434 extensive use of antioxidants. Møller and Mousseau (2007b) have 435 previously reported for birds from Chernobyl that species with longer 436 migrations had lower abundance at high radiation levels. Here we were 437 unable to document a significant main effect of migration on the impact of 438 radiation on abundance. However, we did show a stronger negative impact 439 of radiation on migrants in Chernobyl, but on residents in Fukushima. The 440 Japanese and the European avifaunas differ in their migration distance 441 because more than twice as many species of birds at Fukushima were 442 residents (54% of 57 species) as the species at Chernobyl (21% of 97 443 species). Furthermore, Western Palearctic species migrate longer distances 444 and cross more inhospitable deserts than Eastern Palearctic migrants, while 445 Japanese migrants have the possibility to stop at any of the many 446 archipelagoes during migration. Thus it appears that it is whether a species 447 is migrating or not rather than the actual migration distance that is the 448 important predictor of the effects of radiation on abundance. 449 The slope of the relationship between abundance and radiation was 450 significantly related to whether plumage color was based on carotenoids 451 and pheomelanin. In Fukushima, species without carotenoids in their 452 plumage had lower abundance at high radiation levels, while that was not 453 the case for species with carotenoid-based plumage color. We have 454 previously reported for Chernobyl that species with carotenoid-based 455 plumage are more strongly negatively impacted (Møller and Mousseau 17 456 2007b) as we report here. The slope of the relationship between abundance 457 and background radiation differed between species with and without 458 plumage containing carotenoids and phaeomelanin, but not eumelanin, as 459 we a priori had predicted. The two former kinds of pigment-based 460 coloration impose a trade-off between antioxidant use for maintenance 461 caused by radiation and use of antioxidant pigments allocated to plumage 462 (Møller and Mousseau 2007b; Galván et al. 2011). Interestingly, these 463 effects differed between Fukushima and Chernobyl for carotenoid- and 464 phaeomelanin-based coloration, but not for eumelanin coloration. The main 465 difference between Fukushima and Chernobyl is the duration of chronic 466 exposure to radiation (3 versus 28 years), the duration of accumulation of 467 deleterious mutations (Møller and Mousseau 2011b) and differences in 468 composition of radionuclides (cesium in Fukushima and strontium, cesium 469 and plutonium in Chernobyl). Galván et al. (2014) have previously 470 suggested and provided evidence for pheomelanin playing an active role in 471 resistance to radiation. 472 We have previously investigated the importance of similarity in 473 effect of ionizing radiation on abundance due to common phylogenetic 474 descent, showing that conclusions are independent of phylogeny (Møller 475 and Mousseau 2007b; Galván et al. 2011, 2014). Hence this justifies the 476 absence of phylogenetic analyses in the present study. 477 There may be many causes for differences in population trends of 478 birds. Here we have attempted to control statistically for a number of such 479 factors. We have previously shown that a habitat preference for human 480 proximity and other factors did not account for differences in population 481 trends among species (Møller et al. 2012a, b). 482 In conclusion, we have provided extensive information on the impact 483 of radiation on the abundance of breeding birds at Chernobyl and 484 Fukushima, and the ecological characteristics associated with such impacts 18 485 generally confirmed findings from previous studies at Chernobyl, although 486 most characteristics showed significant differences between Chernobyl and 487 Fukushima. This level of replication suggests that birds may constitute a 488 useful model system for investigating the impact of radiation and the 489 evolution of radiation-resistance in vertebrates. 490 491 Acknowledgments 492 help by Professor A. Hagiwara, T. Kanagawa, K. Kawai, Professor K. 493 Kawatsu and A. M. Smith in Japan. We are especially grateful to Namie- 494 cho and the people of Fukushima Prefecture who permitted and supported 495 us to conduct this study in the field. We gratefully acknowledge support 496 from the US National Science Foundation, the University of South Carolina 497 College of Arts & Sciences, CNRS (France), the Samuel Freeman 498 Charitable Trust, Qiagen GmbH, JSPS KAKENHI Grant Number 499 26440254, and anonymous gifts from individuals in Japan. 500 We gratefully acknowledge logistic support and 19 501 References 502 Asker D, Awad TS, McLandsborough L, Beppu T, Ueda K (2011) 503 Deinococcus depolymerans sp. nov., a gamma- and UV-radiation- 504 resistant bacterium, isolated from a naturally radioactive site. Int J 505 Syst Evol Microbiol 61:1448-1453 506 507 508 509 510 511 512 Beauchamp G, Heeb P (2001) Social foraging and the evolution of white plumage. Evol Ecol Res 3:703–720 Bennett PM, Owens IPF (2002) Evolutionary ecology of birds. Oxford University Press, Oxford, UK Bibby CJ, Hill DA, Burgess ND, Mustoe S (2005) Bird census techniques. Academic Press, London, UK Blondel J, Ferry C, Frochot B (1970) La méthode des indices ponctuels 513 d’abondance (I. P. A.) au des relevés d’avifaune par “stations 514 d’ecoute”. Alauda 38:55-71 515 Bonisoli-Alquati A, Mousseau TA, Møller AP, Caprioli M, Saino N 516 (2010a) Increased oxidative stress in barn swallows from the 517 Chernobyl region. Comp Biochem Physiol A 155:205-210 518 Bonisoli-Alquati A, Voris A, Mousseau TA, Møller AP, Saino N, Wyatt M 519 (2010b) DNA damage in barn swallows (Hirundo rustica) from the 520 Chernobyl region detected by use of the Comet assay. Comp 521 Biochem Physiol C 151:271-277 522 Bump EA, Brown JM (1990) Role of glutathione in the radiation response 523 of mammalian cells in vitro and in vivo. Pharmacol Therapeut 524 47:117-136 525 526 527 528 Cramp S, Simmons KEL (1977-1994) The birds of the Western Palearctic. Oxford University Press, Oxford, UK del Hoyo J, Elliott A, Sagartal J (eds) (1992-2011) Handbook of the birds of the World. Lynx, Barcelona, Spain 20 529 530 531 532 Dighton J, Tugay T, Zhdanova N (2008) Fungi and ionizing radiation from radionuclides. FEMS Microbiol Lett 281:109-120 Draper NR, Smith H (1981) Applied regression analysis. Second edition. John Wiley, New York, NY 533 Galván I (2008) The importance of white on black: unmelanized plumage 534 proportion predicts display complexity in birds. Behav Ecol 535 Sociobiol 63:303-311 536 Galván I, Alonso-Alvarez C (2009) The expression of melanin-based 537 plumage is separately modulated by exogenous oxidative stress and a 538 melanocortin. Proc R Soc B 276:3089-3097 539 Galván I, Bonisoli-Alquati A, Jenkinson S, Ghanem G, Wakamatsu K, 540 Mousseau TA, Møller AP (2014) Chronic exposure to low-dose 541 radiation at Chernobyl favors adaptation to oxidative stress in birds. 542 Funct Ecol 28:1387-1403 543 Galván I, Mousseau TA, Møller AP (2011) Bird population declines due to 544 radiation exposure at Chernobyl are stronger in species with 545 pheomelanin-based coloration. Oecologia 165:827-835 546 Galván I, Solano F (2009) The evolution of eu- and pheomelanic traits may 547 respond to an economy of pigments related to environmental 548 oxidative stress. Pigment Cell Melanoma Res 22:339-342 549 Garamszegi LZ, Møller AP (2010) Effects of sample size and intraspecific 550 variation in phylogenetic comparative studies: A meta-analytic 551 review. Biol Rev 85:797-805 552 Garamszegi LZ, Møller AP (2011) Nonrandom variation in within-species 553 sample size and missing data in phylogenetic comparative studies. 554 Syst Biol 60:676-680 555 Ivaniota L, Dubchak AS, Tyshchenko VK (1998) Free radical oxidation of 556 lipids and antioxidant system of blood in infertile women in a 557 radioactive environment. Ukr Biochim Zh 70:132–135 (in Russian) 21 558 Kryshev I, Alexakhin R, Makhonko K (1992) Radioecological 559 consequences of the Chernobyl accident. Nuclear Society, Moscow, 560 Russia 561 Kryshev II, Ryabov IN (1990) About the efficiency of trophic level in the 562 accumulation of Cs-137 in fish of the Chernobyl NPP cooling pond. 563 In: Ryabov IN, Ryabtsev IA (eds) Biological and radioecological 564 aspects of the consequences of the Chernobyl accident. USSR 565 Academy of Sciences, Moscow, pp 116-121 566 Lislevand T, Figuerola J, Székely T (2007) Avian body sizes in relation to 567 fecundity, mating system, display behavior, and resource sharing. 568 Ecology 88:1605 569 570 571 572 McGraw KJ, Wakamatsu K (2004) Melanin basis of ornamental feather colors in male Zebra Finches. Condor 106:686-690 Møller AP (1983) Methods for monitoring bird populations in the Nordic countries. Nordic Council of Ministers, Oslo, Norway 573 Møller AP, Hagiwara A, Matsui S, Kasahara S, Kawatsu K, Nishiumi I, 574 Suzuki H, Ueda K, Mousseau TA (2012a) Abundance of birds in 575 Fukushima as judged from Chernobyl. Environ Poll 164:36-39 576 Møller AP, Mousseau TA (2007a) Species richness and abundance of birds 577 in relation to radiation at Chernobyl. Biol Lett 3:483-486 578 Møller AP, Mousseau TA (2007b) Determinants of interspecific variation 579 in population declines of birds from exposure to radiation at 580 Chernobyl. J Appl Ecol 44:909-919 581 582 583 584 Møller AP, Mousseau TA (2011a) Efficiency of bio-indicators for lowlevel radiation under field conditions. Ecol Indicat 11:424-430. Møller AP, Mousseau TA (2011b) Conservation consequences of Chernobyl and other nuclear accidents. Biol Cons 144:2787-2798 22 585 Møller AP, Nishiumi I, Suzuki H, Ueda K, Mousseau TA (2012b) 586 Differences in effects of radiation on abundance of animals in 587 Fukushima and Chernobyl. Ecol Indicat 24:75-81 588 Møller AP, Surai PF, Mousseau TA (2005) Antioxidants, radiation and 589 mutation in barn swallows from Chernobyl. Proc R Soc Lond B 590 272:247–253 591 Navarro J, Obrador E, Pellicer JA, Asensi M, Viña J, Estrela JM (1997) 592 Blood glutathione as an index of radiation-induced oxidative stress in 593 mice and humans. Free Radical Biol Med 22:1203-1209 594 595 Neter J, Kutner MH, Nachtsheim CJ, Wasserman W (1996) Applied linear statistical models. Irwin, Chicago, IL 596 Neyfakh EA, Alimbekova AI, Ivanenko GF (1998) Radiation-induced 597 lipoperoxidative stress in children coupled with deficit of essential 598 antioxidants. Biochim (Moscow) 63:977–987 599 Ninni P, de Lope F, Saino N, Haussy C, Møller AP (2004) Antioxidants 600 and condition-dependence of arrival date in a migratory passerine. 601 Oikos 105:55-64 602 Olson V, Owens IPF (2005) Interspecific variation in the use of carotenoid- 603 based coloration in birds: diet, life history and phylogeny. J Evol 604 Biol 18:1534–1546 605 Ozeki H, Ito S, Wakamatsu K, Ishiguro I (1997) Chemical characterization 606 of pheomelanogenesis starting from dihydroxyphenylalanine or 607 tyrosine 608 concentrations and reaction time. Biochim Biophys Acta 1336:539– 609 548 610 611 and cysteine. Effects of tyrosinase and cysteine Riley PA (1994) Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int J Radiat Biol 65:27-33 612 Romanovskaya VA, Rokitko PV, Mikheev AN, Gushcha NI, Malashenko 613 YR, Chernaya NA (2002) The effect of γ-radiation and desiccation 23 614 on the viability of the soil bacteria isolated from the alienated zone. 615 Microbiol 71:608-613 616 SAS Institute Inc (2012) JMP. SAS Institute Inc., Cary, NC 617 Shestopalov VM (1996) Atlas of Chernobyl exclusion zone. Ukrainian 618 619 Academy of Science, Kiev Smith MH, Oleksyk TK, Tsyusko O (2002) Effects of trophic position and 620 ecosystem type on the form of the frequency distribution of 621 radiocesium at Chornobyl and nuclear sites in the United States. In: 622 Proc Int Symp: Transfer of Radionuclides in Biosphere: Prediction 623 and Assessment, December 18-19, 2002, Mito, Japan, pp 37-48 624 Sokal RR, Rohlf FJ (1995) Biometry. W. H. Freeman, New York 625 Tella JL, Figuerola J, Negro JJ, Blanco G, Rodríguez-Estrella R, Forero 626 MG, Blazquez MC, Green AJ, Hiraldo F (2004) Ecological, 627 morphological and phylogenetic correlates of interspecific variation 628 in plasma carotenoid concentration in birds. J Evol Biol 17:156-164 629 Toral GM, Figuerola J, Negro JJ (2008) Multiple ways to become red: 630 Pigment identification in red feathers using spectrometry. Comp 631 Biochem Physiol B 150:147-152 632 Vartanian LS, Gurevich S, Kozachenko AI, Nagler LG, Burlakova EB 633 (1994) Age-related peculiarities of effect of low dose ionizing 634 radiation on blood antioxidant enzyme system status in Chernobyl's 635 accident liquidation participant. Adv Gerontol 14:48-54 (in Russian) 24 636 Voitovich AM, Afonin VYu (2002) DNA damages and radionuclide 637 accumulation in wild small vertebrates. In: Environmental 638 Radioactivity in the Arctic and Antarctic, Proceedings of the 5th 639 International Conference, St. Petersburg, 16-20 June 2002, Russia, 640 pp 340-343 641 Voříšek P, Klvanova A, Wotton S, Gregory RD (2010) A best practice 642 guide for wild bird monitoring schemes. European Union, 643 Bruxelles, Belgium 644 645 646 Wild Bird Society of Japan (1982) A field guide to the birds of Japan. Wild Bird Society of Japan, Tokyo Yakushev BI, Budkevich TA, Zabolotny AI, Mironov V, Kudryashov VP 647 (1999) Contamination of vegetation in Belarus by transuranium 648 radionuclides due to Chernobyl NPP accident. In: Goossens LHJ (ed) 649 Proc 9th Ann Conf “Risk analysis: Facing the new millennium”, 650 October 10-13, 1999. Delft University Press, Rotterdam, pp 841-844 651 652 25 653 Legends to figures 654 655 Fig. 1 Approximate location of census areas around Fukushima, 656 Japan indicated by dark blue dots (A) and Chernobyl, Ukraine and Belarus 657 indicated by light blue dots (B). Note that levels of radiation have only 658 been recorded at a limited number of sites 659 660 Fig. 2 Slope of the relationship between abundance and background 661 radiation in relation to body mass (g) in different species of birds 662 663 Fig. 3 Slope of the relationship between abundance and background 664 radiation in relation to diet in different species of birds 665 666 Fig. 4 Slope of the relationship between abundance and background 667 radiation in relation to migration in different species of birds 668 669 Fig. 5 670 radiation in relation to plumage color based on (A) carotenoids, (B) 671 eumelanin and (C) pheomelanin in different species of birds 672 673 Slope of the relationship between abundance and background 26 674 675 676 677 Fig. 1 27 678 679 680 681 682 Fig. 2 28 683 684 685 Fig. 3 29 686 687 688 689 690 Fig. 4 30 691 692 693 Fig. 5 31 694 32 695 Table 1 Model of slope of the relationship between abundance and level of background radiation in relation to 696 body mass (g), diet (0 – hervivory, 1 – carnivory), migration distance (º latitude), carotenoid score, eumelanin 697 score, pheomelanin score, area (Chernobyl, Fukushima), area x body mass, area x diet, area x migration distance, 698 area x carotenoids, area x eumelanin, area x pheomelanin in a model weighted by sample size. The model had the 699 statistics F = 22.16, df = 13, 140, r2 = 0.64, P < 0.0001 Source df Sum of F P Estimate Error < 0.0001 -0.267 0.050 squares Intercept Body mass 1 17.330 21.85 < 0.0001 0.099 0.021 Diet 1 10.303 10.30 0.0004 -0.045 0.012 Migration distance 1 0.237 0.30 0.59 -0.008 0.014 Carotenoids 1 10.821 13.65 0.0003 0.034 0.009 Eumelanin 1 3.019 3.81 0.053 -0.016 0.008 Pheomelanin 1 0.201 0.25 0.62 0.004 0.007 Area 1 22.620 22.62 < 0.0001 0.073 0.014 Area x Body mass 1 10.361 13.07 0.0004 -0.076 0.021 Area x Diet 1 15.959 20.12 < 0.0001 0.056 0.012 33 700 Area x Migration distance 1 12.148 15.31 < 0.0001 0.055 0.014 Area x Carotenoids 1 25.771 32.50 < 0.0001 -0.052 0.009 Area x Eumelanin 1 0.302 0.38 0.54 -0.016 0.008 Area x Pheomelanin 1 22.583 28.48 < 0.0001 -0.038 0.007 Error 153 111.023 Electronic Supplementary Material Click here to download Electronic Supplementary Material: ESM.doc Authors' Response to Reviewers' Comments Click here to download Authors' Response to Reviewers' Comments: Letter to editor.doc Dear Erik, Thank you for your email. We have made all the changes that you suggested. Please find enclosed this revised version. All the best wishes, Anders